The objectives of the research proposed here are 1) and understanding of the functional organization of the RNA polymerase III-dependent adenoviral VAI promoter, and 2) knowledge of the mechanism of action of the VAI RNA itself. These goals will be approached by an exhaustive genetic analysis of the VAI gene. Point mutations will be produced in vitro by the directed mutagenesis of a cloned VAI gene, and deletion mutations which remove internal segments of the 3' and of the gene will be constructed in vitro from more extensive deletions already available. Genes containing point mutations in the promoter region will then be examined for their ability to support VAI transcription in vitro, and, after being incorporated into viral DNA, in vivo. These studies should permit the construction of a high resolution functional map of the VAI promoter. Genes carrying both point and deletion mutations will be assayed to determine the effects of those mutations on the in vitro binding activities exhibited by wild-type VAI RNA, and will be incorporated into virus to determine what phenotype, if any, they confer in vivo. The data obtained should be useful in the formulation and evaluation of detailed models for VAI RNA function.